Current opinion in structural biology最新文献

While the structure/function paradigm for folded domains was established decades ago, our understanding of how intrinsically disordered regions (IDRs) contribute to biological function is still evolving. IDRs exist as conformational ensembles that can range from highly compact to highly extended depending on their sequence composition. IDR sequences are less conserved than those of folded domains, but often display short, conserved segments termed short linear motifs (SLiMs), that often mediate protein–protein interactions and are often regulated by posttranslational modifications, giving rise to complex functionality when multiple, differently regulated SLiMs are combined. This combinatorial functionality was associated with signaling and regulation soon after IDRs were first recognized as functional elements within proteins. Here, we discuss roles for disorder in proteins that regulate cyclin-dependent kinases, the master timekeepers of the eukaryotic cell cycle. We illustrate the importance of intrinsic flexibility in the transmission of regulatory signals by these entirely disordered proteins.

Evolution has fostered robust DNA damage response (DDR) mechanisms to combat DNA lesions. However, disruptions in this intricate machinery can render cells overly reliant on the remaining functional but often less accurate DNA repair pathways. This increased dependence on error-prone pathways may result in improper repair and the accumulation of mutations, fostering genomic instability and facilitating the uncontrolled cell proliferation characteristic of cancer initiation and progression. Strategies based on the concept of synthetic lethality (SL) leverage the inherent genomic instability of cancer cells by targeting alternative pathways, thereby inducing selective death of cancer cells. This review emphasizes recent advancements in structural investigations of pivotal SL targets. The significant contribution of structure-based methodologies to SL research underscores their potential impact in characterizing the growing number of SL targets, largely due to advances in next-generation sequencing. Harnessing these approaches is essential for advancing the development of precise and personalized SL therapeutic strategies.

RNAs are remarkably versatile molecules that can fold into intricate three-dimensional (3D) structures to perform diverse cellular and viral functions. Despite their biological importance, relatively few RNA 3D structures have been solved, and our understanding of RNA structure–function relationships remains in its infancy. This limitation partly arises from challenges posed by RNA's complex conformational landscape, characterized by structural flexibility, formation of multiple states, and a propensity to misfold. Recently, cryo-electron microscopy (cryo-EM) has emerged as a powerful tool for the visualization of conformationally dynamic RNA-only 3D structures. However, RNA's characteristics continue to pose challenges. We discuss experimental methods developed to overcome these hurdles, including the engineering of modular modifications that facilitate the visualization of small RNAs, improve particle alignment, and validate structural models.

The eukaryotic Mediator, comprising a large Core (cMED) and a dissociable CDK8 kinase module (CKM), functions as a critical coregulator during RNA polymerase II (RNAPII) transcription. cMED recruits RNAPII and facilitates the assembly of the pre-initiation complex (PIC) at promoters. In contrast, CKM prevents RNAPII binding to cMED while simultaneously exerting positive or negative influence on gene transcription through its kinase function. Recent structural studies on cMED and CKM have revealed their intricate architectures and subunit interactions. Here, we explore these structures, providing a comprehensive insight into Mediator (cMED-CKM) architecture and its potential mechanism in regulating RNAPII transcription. Additionally, we discuss the remaining puzzles that require further investigation to fully understand how cMED coordinates with CKM to regulate transcription in various events.

Riboswitches are specialized RNA structures that orchestrate gene expression in response to sensing specific metabolite or ion ligands, mostly in bacteria. Upon ligand binding, these conformationally dynamic RNA motifs undergo structural changes that control critical gene expression processes such as transcription termination and translation initiation, thereby enabling cellular homeostasis and adaptation. Because RNA folds rapidly and co-transcriptionally, riboswitches make use of the low complexity of RNA sequences to adopt alternative, transient conformations on the heels of the transcribing RNA polymerase (RNAP), resulting in kinetic partitioning that defines the regulatory outcome. This review summarizes single molecule microscopy evidence that has begun to unveil a sophisticated network of dynamic, kinetically balanced interactions between riboswitch architecture and the gene expression machinery that, together, integrate diverse cellular signals.

Necroptosis is a lytic form of programmed cell death implicated in inflammatory pathologies, leading to intense interest in the underlying mechanisms and therapeutic prospects. Here, we review our current structural understanding of how the terminal executioner of the pathway, the dead kinase, mixed lineage kinase domain-like (MLKL), is converted from a dormant to killer form by the upstream regulatory kinase, RIPK3. RIPK3-mediated phosphorylation of MLKL's pseudokinase domain toggles a molecular switch that induces dissociation from a cytoplasmic platform, assembly of MLKL oligomers, and trafficking to the plasma membrane, where activated MLKL accumulates and permeabilises the lipid bilayer to induce cell death. We highlight gaps in mechanistic knowledge of MLKL's activation, how mechanisms diverge between species, and the power of modelling in advancing structural insights.

Ion-driven membrane motors, essential across all domains of life, convert a gradient of ions across a membrane into rotational energy, facilitating diverse biological processes including ATP synthesis, substrate transport, and bacterial locomotion. Herein, we highlight recent structural advances in the understanding of two classes of ion-driven membrane motors: rotary ATPases and 5:2 motors. The recent structure of the human F-type ATP synthase is emphasised along with the gained structural insight into clinically relevant mutations. Furthermore, we highlight the diverse roles of 5:2 motors and recent mechanistic understanding gained through the resolution of ions in the structure of a sodium-driven motor, combining insights into potential unifying mechanisms of ion selectivity and rotational torque generation in the context of their function as part of complex biological systems.

Protein kinases are dynamic enzymes that display complex regulatory mechanisms. Although they possess a structurally conserved catalytic domain, significant conformational dynamics are evident both within a single kinase and across different kinases in the kinome. Here, we highlight methods for exploring this conformational space and its dynamics using kinase domains from ABL1 (Abelson kinase), PKA (protein kinase A), AurA (Aurora A), and PYK2 (proline-rich tyrosine kinase 2) as examples. Such experimental approaches combined with AI-driven methods, such as AlphaFold, will yield discoveries about kinase regulation, the catalytic process, substrate specificity, the effect of disease-associated mutations, as well as new opportunities for structure-based drug design.

Cre recombinase is a phage-derived enzyme that has found utility for precise manipulation of DNA sequences. Cre recognizes and recombines pairs of loxP sequences characterized by an inverted repeat and asymmetric spacer. Cre cleaves and religates its DNA targets such that error-prone repair pathways are not required to generate intact DNA products. Major obstacles to broader applications are lack of knowledge of how Cre recognizes its targets, and how its activity is controlled. The picture emerging from high resolution methods is that the dynamic properties of both the enzyme and its DNA target are important determinants of its activity in both sequence recognition and DNA cleavage. Improved understanding of the role of dynamics in the key steps along the pathway of Cre-loxP recombination should significantly advance our ability to both redirect Cre to new sequences and to control its DNA cleavage activity in the test tube and in cells.

The rapid advancement in computational power available for research offers to bring not only quantitative improvements, but also qualitative changes in the field of biomolecular simulation. Here, we review the state of biomolecular dynamics simulations at the threshold to exascale resources becoming available. Both developments in parallel and distributed computing will be discussed, providing a perspective on the state of the art of both. A main focus will be on obtaining binding and conformational free energies, with an outlook to macromolecular complexes and (sub)cellular assemblies.